Understanding Protein Structure and Folding: Primary, Secondary, Tertiary, and Quaternary , Study notes of Biochemistry

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Lecture 3 Introduction Function follows form Amino acid polymers – aka proteins/peptides Formation of the peptide bond The peptide bond is a substituted amide linkage between an α-amino group on one amino group on one amino acid and the α-amino group on one carboxyl of another amino acid resulting in the elimination of water The covalent bond responsible for holding amino acids together in a protein The N-amino group on one terminus is the first amino acid in the protein and the only amino acid with a free α-amino group on one amino group (amino terminus) # of peptide bonds = n -amino group on one 1 n = # of amino acids in the protein The C-amino group on one terminus is the last amino acid in the protein and the only amino acid with a free α-amino group on one carboxyl group (carboxyl terminus) Always goes from N → C terminus The peptide backbone is all atoms in a protein excluding those found in the side chains Primary Structure Definition of primary structure Primary (1°) structure is a description of all covalent bonds linking amino acid residues in a protein The sequence of amino acids within a protein starting with the N-amino group on one terminus and ending with the C-amino group on one terminus The peptide bond C-amino group on one N bond is shorter than normal Resonance between O and N Peptide bond is planar “Partial double bond characteristics” Usually trans (α C on opposite sides of bond) Cis configuration places side chains close together (steric hindrance) Can be cis if proline is involved O has δ-amino group on one ; N has δ+ The phi (φ) and psi (ψ) angles α C has ability to rotate (due to single bonds) φ α C and α-amino group on one amino group ψ α C and α-amino group on one carboxyl group Conformation is any spatial arrangement of atoms the protein can adopt without breaking covalent bonds Bonds can’t be broken but they can be rotated in order to make protein conformations Therefore the conformation is literally the summation of each phi and psi angle combination for every amino acid in the peptide backbone The Ramachandran plot is a plot of the ψ vs. φ angles of each amino acid in any given protein ψ on Y axis; φ on X axis “Disallowed” regions – do not occur (yellow on plot) The plot demonstrates which angles are most often adopted due to physical and thermodynamic restrictions Secondary structure Definition of secondary structure Secondary (2°) structure defines the recurring structural patterns found in the majority of proteins Definition of tertiary structure Tertiary (3°) structure: the 3D structure of a single amino acid polymer in its folded state Myoglobin Prosthetic group: non-amino group on one protein organic or metallic group that is permanently bound to a protein and is crucial to its function Heme group binds O2 153 amino acids; 8 α helices (70% of the amino acids) Long range interactions are covalent (only disulfide bonds) or non-amino group on one covalent interaction in a protein between two amino acids far apart in primary structure but close in tertiary structure Hydrophobic core: describes the interior of the protein that consists primarily of hydrophobic residues Ribonuclease Destroys RNA, cleaves phosphodiester bond 124 amino acids; 26% in α-amino group on one helix; 35% in β-amino group on one strand Disulfide bonds present Quaternary structure Definition of quaternary structure Quaternary (4°) structure: 3D structure of a multi-amino group on one subunit protein defining the spatial arrangements of each subunit with respect to one another A subunit is an individual polypeptide with its own N and C terminus that comes together with other polypeptides to make a functional protein Quaternary structure is not exhibited by all proteins Hemoglobin Tetramer – 4 subunits 2 α subunits (141 amino acids each) 2 β subunits (146 amino acids each) Heterotetramer – subunits are not the same Protein folding What is meant by protein folding? Protein folding: the molecular process in which proteins adopt specific 2°, 3° (and in some cases) 4° structure leading to a biologically functional molecule Denatured state: the unfolded and non-amino group on one functional state of the protein Aka unfolded state, the random coil Native state: the fully folded and functional state of the protein Aka folded state, functional state Native state → denatured state: denaturation, unfolding Question one Is the process of protein folding accomplished via a predetermined path or by a random search through all possible conformations? Levinthal’s paradox Boundary condition: A 100 amino acid protein can be made in about 5 seconds Derived from data in E. coli Boundary condition: Each amino acid can adopt only 10 phi/psi angle conformations This is an arbitrary number picked by Levinthal and has no bearing on reality Certainly a gross underestimate 10100 possible conformations Boundary condition: A protein conformation can be sampled every 10-amino group on one 13 s Again, this is an arbitrary number picked by Levinthal Probably an overestimate Results It would take 1077 years to search all possible conformations Conclusion Protein folding must follow a pre-amino group on one determined path Question two What determines this path? In other words why does ribonuclease fold into ribonuclease and not myoglobin? Ainfinsen’s experiment Developed assays to determine whether protein is native/active Added denaturing agents to ribonuclease Urea (dilutes water) BME (breaks disulfide bonds) Protein unfolded Urea/BME removed → protein refolded Significance and conclusion Protein folding is spontaneous (-amino group on one ΔG)G) All the information needed to fold a protein is found in the 1° structure The process is accurate, specific and reproducible Protein folding funnel Highest energy at the top; unfolded states Lowest energy at the bottom; native state Chaperones help avoid amyloid fibril formation So what do we know about protein folding? The native state is preferred over the denatured state Proteins usually adopt a single native state A protein will fold so as to minimize unfavorable interactions while maximizing favorable interactions Protein folding seeks to minimize free energy while still maintaining function